摘要
目的构建含有绿色荧光蛋白基因(green fluorescent protein gene,gfp)的质粒,电转化从鸡胃肠中分离的乳酸杆菌,并口服接种小鸡表达绿色荧光蛋白(green fluorescent protein,GFP)的重组乳酸杆菌,观察该乳酸杆菌在胃肠道中的分布。方法在获得稳定表达绿色荧光蛋白的德氏乳杆菌乳亚种D17(D17-GFP)菌株后,口服接种20日龄的鸡,于接种后1.5、6、12、24、48和72h分别取胃肠内容物接种培养并计数,同时取胃、空肠、回肠、盲肠、肝和脾脏做冰冻切片在荧光显微镜下观察D17-GFP的分布。结果D17-GFP在接种鸡6h后,胃肠道的内容物中可发现D17-GFP,在42h时细菌数达到最大,并且3d后菌量仍维持在1×104CFU/g左右。在荧光显微镜下发现D17-GFP主要存在于肠道的内容物和黏液中。结论GFP在德氏乳杆菌乳亚种D17中得到了稳定的表达,重组D17-GFP可在鸡胃肠道的黏膜处定植。
Objective To investigate the distribution of the recombinant Lactobacillus delbrueckii subspecies lactis containing green flu- orescent protein gene （gfp） in gastrointestinal （GI） tract of chicken. Methods The plasmid encoding the gfp gene was constructed and electroporated into lactobecilli which were isolated from GI tract of chicken. Chicken of 20 days old were orally inoculated with the recombinant continual expressing strain Lactobecillus delbmeckii subspecies lactis D17 （D17-GFP）. At 1.5, 6, 12, 24, 48, and 72 h postadministration, the animals were sacrificed respectively. Luminal contents from the GI tract were serially diluted in saline and cultured on MRS plates. The sections （12 ram） of stomach, jejunum, ileum, caecum, liver, and spleen were respectively examined with an Olympus fluorescence microscope. Re- suits Six hours after ingestion, D17-GFP was detectable in all samples. At 42 h postadministration, the microorganism was present throughout the intestine （stomach, jejunum, ileum, and caecum） with maximum concentrations. Certain lactobecilll （1 ×10^4 CFU/g） were still persisting in the GI tract after 3 d. ~ fluorescem microscopy indicated that the D17-GFP was mainly localized in the luminal contents and mucus of the intestine. Conclusion GFP is expressed in D17 stably and the recombinant lactobecillus expressing GFP could settle down at the mncosa of GI tract.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第2期126-130,共5页
Immunological Journal
基金
"973"项目资助(2004CB117505)
关键词
乳酸杆菌
绿色荧光蛋白
胃肠道
分布
Lactobacilli
Green fluorescent protein gene
Gastrointestinal tract
Distribution
